The fungus Cryptococcus neoformans is a respiratory pathogen that is a relatively frequent cause of life-threatening disease in individuals with impaired immunity. Macrophages are critical cells in the control and dissemination of infection in the lung. C. neoformans is a facultative intracellular pathogen that can replicate in macrophage after damaging the phagolysosomal membrane. Fungal damage to the phagolysosomal membrane determines the outcome of the fungal-macrophage interaction. For C. neoformans, as well as other soil pathogenic fungi, the capacity for virulence has been proposed to emerge from interactions with phagocytic predators in the environment that select for traits that can then promote virulence in mammals. In fact, one of the remarkable aspects of C. neoformans pathogenesis is that it is virulent in many different animal hosts and can replicate in phagocytic cells from mammals, fish, insects and protozoa. This poses the fascinating question of how a soil organism with no need for an animal host is able to subvert immune effector cells from so many species. We hypothesize that this capacity for non-specific virulence comes from its ability to undermine host cells with an ?intracellular pathogenesis kit? that includes the intracellular secretion of numerous effector molecules. We have preliminary data that C. neoformans releases many proteins inside murine macrophages that include several well-known virulence factors. In this application we propose to identify proteins made inside human macrophages and amoeba that undermine host cells. A subset of these proteins will then be studied in detail to dissect mechanisms of intracellular pathogenesis. Three specific aims are proposed: Aim 1. To identify CN proteins produced intracellularly in human macrophages; Aim 2. To identify key CN compounds produced during its interaction with amoeba; Aim 3. To ascertain the mechanism of action of certain CN proteins in the intracellular pathogenic strategy in phagocytic human and protozoan cells. This work will illuminate the mechanisms of C. neoformans intracellular pathogenesis and this information can be relevant to designing improved therapies, antifungal therapies and vaccines.